Assessement of a possible relation between

osteoporosis and hypertension in

spontaneously hypertensive rats and
recombinant inbred strains.
Rana El Bikai1, Pierre Dumas1, Junzheng Peng1, Svetlana Koltsova1, Nathalie Dion1, LouisGeorges Ste-Marie1, Vladimir Ken2, Drahomira Kenova2, Michal Pravenec3, Ondrej eda1,
Johanne Tremblay1,Pavel Hamet1.
1

CHUM Research Center, (CRCHUM), Qubec, Montral, Canada

1st Medical Faculty, Charles University, Prague, Czech Republic
3
Institute of Physiology, Prague, Czech Republic
2

Aims:
Osteoporosis and hypertension are two age related diseases that could be
associated with accelerated aging. Our project focuses on studying the common
genetic determinants related with the progression of hypertension and the changes in
bone density and the bone structural parameters associated to osteoporosis.
Methods:
The spontaneously hypertensive rats (SHR) exhibit a gradual increase in blood
pressure during puberty and young adulthood that resembles what is seen in human
essential hypertension. It thus makes it a good genetic model for studying
hypertension and its complications (1). In addition to high blood pressure, several
abnormalities in metabolic pathways have been reported in the SHR. Many of these
defects are related to calcium handling and several groups have reported abnormal
bone mineralization (2,3). Hence, SHR is also a good model to study osteoporosis.
Recombinant inbred strains (RIS) are inbred replica of F2 segregating population
obtained after several round of brother-sister mating (>20) of progeny of fixed
(selected)
F2
pairs
(Figure
1).
As
such,
RIS
are
a unique opportunity to study genetically identical animals at different
ages, and are considered as a very valuable tool for genetic studies since the
mapping of their genome is practically permanent and they allow longitudinal studies
(they are inbred strains) (4). They possess 50% of each of the two original ancestral
genomes but in different combinations. This allows the dissection and mapping of the
polygenic phenotypes segregating in the RIS along with the various combinations of
the two progenitor genomes. One of the largest set of RIS for study of cardiovascular
and metabolic traits was developed by the crossing of two genetically distant parental
strains: the SHR and the normotensive Brown Norway rat (BN-Lx). Reciprocal
crosses of SHR and BN-Lx are represented as follows: HxB and BxH (H representing
SHR and B representing BN-Lx). The subsequent inbreeding of rats for over 35
generations has produced the strains available today and the present work was
performed using these HxB/BxH RIS (5-9). Using those strains we have proposed
that hypertension is a case of accelerated aging. Here we are reporting initial studies

of another aging related phenotype, osteoporotic bone changes as a parallel of

pleotropic characteristic of hypertension (8). In Study 1, we assessed major bone
structure characteristics to initiate exploration of strain distribution pattern: BxH9,
BxH11, BxH2, HxB23 and HxB3. The bone mineral density (BMD) was measured by
DXA and histomorphometric analysis was performed on cancellous bone of
decalcified L4 vertebra. Bones were obtained from male rats of fifteen weeks of age
and kept in 70 % ethanol. Statistical analysis was done using one way ANOVA.
As the onset of osteoporosis and hypertension is gradual and both conditions evolve
over time, in Study 2 we assessed the intra-strain temporal dynamics of blood
pressure (assessed by telemetry) and bone parameters (microCT). Therefore, we
studied 3 different RIS (HxB3, HxB13 and HxB17) and the SHR parental strain which
served as a positive control for hypertension and bone disorders. This longitudinal
study was performed over a year so that we could follow the growth of rats and
assess the possible morphological changes at different ages. Systolic, diastolic and
mean blood pressures as well as heart rate were assessed by telemetry
measurements at 3, 6, 9 and 12 months of age. Radiotelemetry transducers (Data
Science International, St.Paul, MN) were placed in the lower abdominal cavity and
connected to catheters implanted in the lower abdominal aorta of anesthetized rats
under aseptic conditions following standard operating procedures at the CRCHUM.
The measurements were performed after two weeks of recovery. The structural
parameters of the left tibial bone were obtained from in vivo microCT scans (skyscan
1076: Skyscan, Antwerp, Belgium) at the four different ages (3, 6, 9 and 12 months)
in the same animals to assess the morphological changes. Three-dimensional
analysis was then performed following the standard bone package analysis
developed at Skyscan.
Results
In study 1, HxB3 strain showed a higher BMD then BxH9 (p<0.01), BxH11 (p<0.001)
and BxH2 (p<0.001), and a greater trabecular bone volume (BV/TV) then BxH9
(p<0.001) and BxH11 (p<0.001). Resorption parameters showed that eroded
surfaces (ES/BS) were significantly lower in HxB3 compared with the other groups
(vs BxH9 p<0.01; vs BxH11, BxH2 and BxH23, p<0.001). The number of osteoclasts
per bone perimeter (N.Oc/Bpm) was significantly lower in HxB3 compared with BxH9
(p<0.01) (Figure 2). Finally, the results showed that HxB3, compared to the other
RIS, possesses genetic factors which have positive effects on trabecular bone mass,
possibly by lowering bone resorption.
In study 2, the results showed that HxB17 rats have extreme phenotypes as
compared to the other strains. For instance, highest diastolic blood pressure and
systolic blood pressure detected in the RIS was HxB17 (p<0.0001) (Figure 3). In
addition, our scans showed that HxB17 rats have more trabecular weaknesses than
the other strains for example the trabecular thickness was the lowest in HxB17
(p<0.0001) compared to the other strains. And the HxB 17 showed the lower
trabecular bone volume (BV/TV) (p<0.0001) then the other rats (Figure 4).

Conclusion
The data from study 1 indicated the existence of strain differences in bone
parameters, from which we conclude that the HxB3 has the most robust bone of all

the tested strains. The observed differences in BV/TV and resorption parameters
between the tested RIS are large enough to potentially enable us to determine the
loci related to these parameters of bone mass and remodelling (analysis is
underway). The strain distribution pattern of the traits will then serve to map the
genetic loci responsible for the observed differences, using the publicly available
genetic map of all HXB BXH RIS containing over > 20,000 single nucleotide
polymorphisms. In the second study, we observed that HxB17 shows extreme
phenotypes compared to the other RIS, displaying weak trabecular architecture and
high blood pressure.
Taken together, our data unveiled two strains, HxB3 and HxB17 which display
opposite phenotypes. Fine mapping and identification of causal polymorphisms
participating in the genetic architecture responsible for the observed bone density
and blood pressure-related traits will be achieved by combination of genetic
(advanced intercross lines) and bioinformatic (genetical genomics, network analysis)
approaches.

Figures

Figure 1: The construction of the recombinant inbred strain panel (SHRxBN-Lx).

RI strain Age
(week)

BMD
L1-L4 BV/TV
(g/cm2)
(%)

ES/BS (%)

N.OC/BPm
(mm)

BxH9,
n=3

15

0.183 0.009

16.8 1.7 10.7 0.3

0.97 0.13

BxH11,
n=4

15

0.178 0.007

17.5 4.5 12.2 2.6

0.66 0.29

0.173 0.01

25.5 5.2 11.3 0.8

0.56 0.21

BxH2, n= 15
3
HxB23,
n=3

16

0.203 0.012

25.4 4.6 17.1 0.5

0.61 0.35

HxB3,
n=5

15

0.212 0.002

31.9 1.4 6.4 1

0.29 0.03

Figure 2: Bone parameters of five recombinant inbred strains of male rats at fifteen
weeks of age assessed by DXA. (BMD = Bone Mineral Density, BV/TV = Bone
Volume/ Tissue Volume, ES/BS = Eroded Surface/Bone Surface, N.OC/BPm =
Number of osteoclast per bone primeter)

Figure 3: Diastolic, systolic, mean arterial pressure and heart rate in SHR rats and
recombinant inbred strains (HxB3, HxB23 and HxB17).

Trabecular Thickness

BV/TV

1.0

0.4

HxB3
HxB13
HxB17
SHR
%

0.8

mm

0.6

HxB3
HxB13
HxB17
SHR

0.3
0.2

0.4
0.1

0.2
0.0

0.0
3

Month

12

12

Month

Figure 4: Trabecular thickness and bone trabecular bone volume ratio in HxB3,
HxB13, HxB17 and SHR rats.

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Supported by CIHR grant:

Hypertension at nexus of thrifty, thirsty and accelerated ageing genotypes: and
evolution of complex disease.